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Biomedical subjects

J Kusari

Publications and source records attributed to J Kusari.

33 records · Page 2Linked to original sources
PubMed1991

Insulin resistance and diabetes due to different mutations in the tyrosine kinase domain of both insulin receptor gene alleles.

Mutations in the insulin receptor gene can lead to in vivo and in vitro insulin resistance and can be the cause of diabetes mellitus in selected patients. We have studied a 22-year-old diabetic woman with Type A insulin resistance and acanthosis nigricans. Insulin binding to the patient's erythrocytes, monocytes, adipocytes, fibroblasts, and transformed lymphocytes was decreased. Receptor autophosphorylation and tyrosine kinase activity toward an exogenous substrate were reduced in partially purified insulin receptors from the proband's transformed lymphocytes. Determination of the nucleotide sequence of the patient's insulin receptor cDNA revealed that the subject was a compound heterozygote who inherited two different mutant insulin receptor gene alleles. The paternal allele contains a missense mutation encoding the substitution of glutamine for arginine at position 981 in the tyrosine kinase domain of the receptor. The maternal allele contains a nonsense mutation causing premature termination after amino acid 988 in the beta-subunit, thereby deleting most of the kinase domain. The mRNA encoded by the allele with the premature stop codon is likely to be unstable, since mRNA transcripts from this allele were decreased markedly compared with the other allele. The mother, who is heterozygous for the nonsense mutation, exhibited only mild insulin resistance, whereas the proband was severely insulin-resistant; this indicates that the missense mutation is biologically significant. In summary, (1) we have identified a patient and her family with a genetic form of insulin resistance and diabetes due to a defect at the level of the insulin receptor; (2) the proband is a compound heterozygote displaying a missense mutation (position 981) in one allele and a nonsense mutation (position 988) in the other insulin receptor gene allele; (3) the missense mutation is in the kinase domain and encodes a receptor with impaired in vitro kinase activity; and (4) based on the in vitro and in vivo phenotype, the kinase domain mutation at position 981 is biologically significant leading to insulin resistance.

Adult↗
PubMed1991

Analysis of the gene sequences of the insulin receptor and the insulin-sensitive glucose transporter (GLUT-4) in patients with common-type non-insulin-dependent diabetes mellitus.

Insulin resistance is a common feature of non-insulin-dependent diabetes mellitus (NIDDM) and "diabetes susceptibility genes" may be involved in this abnormality. Two potential candidate genes are the insulin receptor (IR) and the insulin-sensitive glucose transporter (GLUT-4). To elucidate whether structural defects in the IR and/or GLUT-4 could be a primary cause of insulin resistance in NIDDM, we have sequenced the entire coding region of the GLUT-4 gene from DNA of six NIDDM patients. Since binding properties of the IRs from NIDDM subjects are normal, we also analyzed the sequence of exons 16-22 (encoding the entire cytoplasmic domain of the IR) of the IR gene from the same six patients. When compared with the normal IR sequence, no difference was found in the predicted amino acid sequence of the IR cytoplasmic domain derived from the NIDDM patients. Sequence analysis of the GLUT-4 gene revealed that one patient was heterozygous for a mutation in which isoleucine (ATC) was substituted for valine (GTC) at position 383. Consequently, the GLUT-4 sequence at position 383 was determined in 24 additional NIDDM patients and 30 nondiabetic controls and all showed only the normal sequence. From these studies, we conclude that the insulin resistance seen in the great majority of subjects with the common form of NIDDM is not due to genetic variation in the coding sequence of the IR beta subunit, nor to any single mutation in the GLUT-4 gene. Possibly, a subpopulation of NIDDM patients exists displaying variation in the GLUT-4 gene.

Aged↗
PubMed1991

Insulin-receptor cDNA sequence in NIDDM patient homozygous for insulin-receptor gene RFLP.

Resistance to insulin action is a well-established feature of non-insulin-dependent diabetes mellitus (NIDDM) and is believed to contribute to the etiology of this condition. A strong genetic contribution to the etiology of NIDDM exists, and we previously identified an insulin-receptor gene restriction-fragment-length polymorphism (RFLP) associated with the NIDDM phenotype. In an attempt to elucidate whether structural defects in the insulin receptor could be a primary cause of insulin resistance in NIDDM, we analyzed the insulin-receptor cDNA sequence in a subject with NIDDM who is also homozygous for this RFLP. The insulin-receptor cDNA was sequenced with the polymerase chain reaction (PCR). mRNA from transformed lymphocytes was reverse transcribed and amplified with five overlapping sets of primers that span the coding sequence of both alpha- and beta-subunits. No difference was found in the predicted amino acid sequence of the subject's insulin receptor compared with the normal insulin receptor. At nucleotide positions 831 and 2247, the subject is heterozygous for silent nucleotide polymorphisms that do not affect the amino acid sequence. Exon 11 encodes a 12-amino acid insert in the alpha-subunit, which, due to alternate splicing, is not expressed in lymphocyte insulin-receptor mRNA. Consequently, exon 11 was amplified from genomic DNA by PCR; the sequence of exon 11 was found to be normal. In addition, when this patient's transformed lymphocytes were maintained in culture, no abnormalities in insulin binding were observed. We conclude that the insulin resistance seen in this NIDDM subject is not due to a structural alteration in the insulin receptor itself.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Structure of testicular angiotensin-converting enzyme. A segmental mosaic isozyme.

The complete amino acid sequence of rabbit testicular angiotensin-converting enzyme has been deduced from the sequence of the corresponding cDNA clone. A protein of the expected molecular weight of 84,000 was translated in vitro from the mRNA encoded by this cDNA. All of the previously determined sequences of seven tryptic peptides from the enzyme are present in the deduced sequence, thus confirming the identity of the protein. From the deduced sequence it appears that the protein contains a signal peptide at the amino terminus and a hydrophobic anchoring domain near the carboxyl terminus. Northern analysis with oligonucleotide probes, whose sequences represented different regions of the cDNA, revealed not only the regions of extensive homology between the mRNAs encoding the testicular and the pulmonary isozymes but also a stretch of sequence near the 5' end unique to the testicular mRNA.

Amino Acid Sequence↗
PubMed1988

Isolation of cDNA clones of rabbit angiotensin converting enzyme: identification of two distinct mRNAs for the pulmonary and the testicular isozymes.

We have isolated cDNA clones of rabbit angiotensin converting enzyme. These clones were isolated by antibody-screening of a lambda gt11 expression library made from rabbit testicular mRNA. The 2.6 kb insert of one such clone was subcloned in pBR322 and used as a hybridization probe. Out of the twenty independently isolated clones only seven hybridized with this probe suggesting that these clones belong to at least two families. Northern analysis revealed the presence of a 2.6 kb mRNA in rabbit testes and a 5.0 kb mRNA in rabbit lungs which hybridized strongly with this probe. These results indicate that the two tissue-specific isozymic forms of angiotensin converting enzyme are encoded by two distinct mRNAs which share sequence homologies.

Animals↗
PubMed1988

Gene induction by interferons and double-stranded RNA: selective inhibition by 2-aminopurine.

Transcription of several interferon-inducible human genes is also induced by double-stranded RNA. The nature and the mechanism of action of signals generated by interferons and by double-stranded RNA which mediate the induction of these genes are under investigation. Here we report that 2-aminopurine, a known inhibitor of protein kinases, could selectively block this induction process. Induction of mRNAs 561 and 6-16 in HeLaM cells by double-stranded RNA was completely inhibited by 10 mM 2-aminopurine, whereas cellular protein and RNA syntheses as well as the induction of metallothionein mRNA by CdCl2 were unaffected by this inhibitor. In addition, 2-aminopurine blocked the induction of the same two mRNAs and of mRNAs 2-5(A) synthetase, 2A, and 1-8 by alpha interferon and of mRNAs 2A and 1-8 by gamma interferon in HeLaM cells. The observed inhibition was at the level of transcription, and for establishing efficient inhibition, the 2-aminopurine treatment had to begin at early stages of interferon treatment. In GM2767 cells, 2-aminopurine inhibited induction of mRNAs 561 and 6-16 by double-stranded RNA but not by alpha interferon. These results suggest that double-stranded RNA-induced signal 2 is distinct from the interferon-alpha-induced signal 2 (R. K. Tiwari, J. Kusari, and G. C. Sen, EMBO J. 6:3373-3378, 1987) and that 2-aminopurine can block the former but not the latter. Moreover, it appeared that 2-aminopurine could block the production of signal 1 by interferons. This was confirmed by experiments in which we separately tested the effects of 2-aminopurine on signal 1 and signal 2 production by interferons in HeLaM cells. Although no direct experimental evidence is available as yet, our results are consistent with the hypothesis that the functioning of a protein kinase activity may be necessary for transcriptional induction of genes by double-stranded RNA and for gene induction by interferons in those cells in which signal 1 production is needed.

2-Aminopurine↗
PubMed1987

Functional equivalents of interferon-mediated signals needed for induction of an mRNA can be generated by double-stranded RNA and growth factors.

In our earlier studies we demonstrated that in HeLaM cells, interferon-alpha produces two functionally distinguishable signals, both of which are needed for induced transcription of mRNA 561 and other inducible mRNAs. Interferon-gamma cannot induce mRNA 561 because it produces only signal 1. Here we report that platelet-derived growth factor or epidermal growth factor could also produce signal 1. On the other hand, signal 2, which can be produced by interferon-alpha but not by interferon-gamma, could be elicited also by double-stranded RNA. Several lines of evidence suggest that the production of signal 2 by double-stranded RNA was not mediated through interferon. Interferon-induced transcription of mRNA 561 in HeLaM cells or in human fibroblast GM2767 cells was transient. However, in interferon-alpha-treated GM2767 cells, which had ceased to synthesize mRNA 561, transcription of this mRNA could be induced effectively by double-stranded RNA suggesting that this induction process could bypass the interferon-mediated down-regulation of induced transcription. Unlike HeLaM and GM2767 cells, in Daudi cells, induction of mRNA 561 by interferon-alpha was not transient. Transcription of this and two other induced mRNAs continued at a high rate even after 18 h of interferon-alpha treatment of these cells. The lack of down-regulation of interferon-induced gene expression may be responsible for interferon's acute antigrowth effects on these cells.

Cell Line↗
PubMed1987

Chromosomal localization of the interferon-inducible human gene encoding mRNA 561.

Treatment of human cells with interferon-alpha transiently increases the rate of transcription of mRNA 561, which encodes a 56-kD protein of unknown function. Using a cDNA probe specific for this mRNA, we determined the chromosomal localization of the corresponding gene. Southern blot analyses of genomic DNA samples isolated from 19 independent mouse-human and hamster-human cell hybrids, each of which contained all rodent chromosomes and several human chromosomes, indicated that this gene is located on human chromosome 10. The same conclusion was drawn independently from studies using in situ hybridization of the probe to human metaphase chromosomes. Furthermore, the latter approach enabled us to conclude that the gene is situated most likely at the junction of 10q25-q26.

Animals↗
PubMed1987

Expression of interferon-inducible genes in RD-114 cells.

RD-114 is a cell line which is partially responsive to interferon (IFN). Although both IFN-alpha and IFN gamma inhibit production of the resident retrovirus, they do not inhibit replication of other viruses, such as vesicular stomatitis virus and encephalomyocarditis virus, in these cells. In the studies reported here, we studied the characteristics of induction of seven IFN-inducible mRNAs in RD-114 cells. We observed that mRNAs 561, 6-16, 1-8, 2A, and 6-26 have similar induction characteristics in RD-114 cells and in HeLa cells, a fully responsive line. mRNA 2'-5'-oligo-adenylate synthetase (2-5(A) synthetase), however, was induced more efficiently by IFN-alpha in HeLa cells than in RD-114 cells. The same was true for the induction of metallothionein II mRNA by IFN-gamma. However, the latter mRNA was induced equally strongly in both lines when ZnCl2 was used as the inducer, suggesting that the gene is not defective in RD-114 cells. Although IFN-alpha induced 2-5(A) synthetase mRNA poorly and IFN-gamma did not induce it at all in these cells, a mixture of IFN-alpha and IFN-gamma induced this mRNA quite effectively, to a level of induction comparable to that in HeLa cells. Only 1 U of IFN-gamma per ml was sufficient to elicit this synergism, and the data suggested that an IFN-gamma-inducible protein was needed for this process. Induction of mRNA 561 by IFN-alpha in RD-114 cells, unlike that in HeLa cells, did not need ongoing protein synthesis. Once induced, this mRNA turned over rapidly in both cell lines, and this turnover could be slowed down by inhibiting protein synthesis in either cell line. IFN-induced mRNAs, such as 561 and 1-8, were polysome associated in IFN-treated RD-114 cells, suggesting that they were actively translated. Therefore, it is unlikely that the products of these IFN-inducible genes, by themselves, mediate the inhibition of replication of those viruses which are insensitive to IFN action in RD-114 cells.

2',5'-Oligoadenylate Synthetase↗
PubMed1987

Clonal derivatives of the RD-114 cell line differ in their antiviral and gene-inducing responses to interferons.

The human rhabdomyosarcoma cell line RD-114 is partially responsive to interferons (IFNs). In these cells, alpha interferon (IFN-alpha) or gamma interferon (IFN-gamma) inhibits the replication of some viruses but not of others. Similarly, some of the IFN-inducible mRNAs are induced poorly, whereas others are induced well. Here we report the isolation of clonal derivatives of this line which display different spectra of responses to IFNs. Among the eight extensively characterized clonal lines, one, C10, did not respond to IFN-alpha or IFN-gamma at all. Retrovirus production by each of the seven other lines was inhibited by both IFN-alpha and IFN-gamma. Replication of vesicular stomatitis virus was inhibited strongly by IFN-alpha in clone B1 but not in others, whereas it was not appreciably affected by IFN-gamma in any clone. Replication of encephalomyocarditis virus was inhibited strongly by IFN-gamma in clones A1, A2, A3, B3, and B8 and by IFN-alpha in clone A2. Neither IFN inhibited the multiplication of these clones greatly, although their doubling times were slightly increased. Five mRNAs were induced by IFNs to varying degrees in the seven clones. mRNA 2A was most strongly induced by IFN-gamma in clone A3. mRNA 1-8 was strongly induced by IFN-alpha in clone A1 and by either IFN in clones A2 and A3. The highest concentrations of 2',5'-oligoadenylate synthetase mRNA, mRNA 561, and mRNA 6-16 were in IFN-alpha-treated clones A1 and A2. These results demonstrated the existence of clonal heterogeneity in IFN responses in a cell line and strengthened the view that IFN treatment of cells generates multiple signals leading to a variety of IFN-induced phenotypes.

Acetyltransferases↗
PubMed1987

Transcriptional analyses of interferon-inducible mRNAs.

Using nuclear runoff transcription assays we demonstrated that alpha interferon-mediated induction of transcription of four mRNAs in HeLa monolayer cells needed ongoing protein synthesis and that such a need could be obviated by pretreating the cells with gamma interferon which, by itself, did not induce transcription of these mRNAs. In another human cell line, RD-114, synthesis of alpha interferon-inducible mRNA 561 did not need ongoing protein synthesis. In this line, however, in which interferon inhibits replication of some viruses but not of others, transcription of two of the six interferon-inducible mRNAs that we examined was not appreciably enhanced by interferon.

HeLa Cells↗
PubMed1986

Regulation of synthesis and turnover of an interferon-inducible mRNA.

Regulation of synthesis and turnover of an interferon (IFN)-inducible mRNA, mRNA 561, in HeLa monolayer cells was studied. Cytoplasmic levels of this mRNA were estimated by hybridization analyses with a cDNA clone that we have isolated as a probe. IFN-alpha A induced a high level of this mRNA in a transient fashion, whereas no induction was observed in response to IFN-gamma. Surprisingly little mRNA 561 was induced in cells treated simultaneously with IFN-alpha A and an inhibitor of protein synthesis, suggesting that in addition to IFN-alpha A, an interferon-inducible protein was needed for induction of this mRNA. Apparently this putative protein could be induced by IFN-gamma as well. Thus, although little mRNA 561 was synthesized in cells treated either with IFN-gamma alone or with IFN-alpha A and cycloheximide, a large quantity of this mRNA was induced in cells which had been pretreated with IFN-gamma and then treated with IFN-alpha A and cycloheximide. Once mRNA 561 was induced by IFN-alpha A, it turned over rapidly. This rapid turnover could be blocked by actinomycin D or cycloheximide indicating that another IFN-inducible protein may mediate this process.

Cell Adhesion↗
PubMed1986

Molecular and genetic comparisons of two serotypes of epizootic hemorrhagic disease of deer virus.

The virus-specific double-stranded genome RNA of 2 serotypes of epizootic hemorrhagic disease of deer virus (EHDV) was evaluated by use of coelectrophoresis in polyacrylamide and agarose gel systems. The molecular weights of virion RNA segments were 0.32 to 2.57 X 10(6) for EHDV-1 and 0.33 to 2.54 X 10(6) for EHDV-2. Seven of 10 double-stranded RNA segments of the 2 serotypes had different electrophoretic mobilities in the polyacrylamide-gel electrophoresis system. Although the individual RNA segments of each serotype contained unique RNA sequences determined on the basis of 2-dimensional polyacrylamide-gel electrophoresis analysis of oligonucleotides, the corresponding segments of the 2 serotypes were found to be comparable and at least 1 pair of RNA segment was almost identical. Virus-specific polypeptides for the 2 serotypes were compared by use of gel electrophoresis. Eleven polypeptides were detected for EHDV-1 and 10 for EHDV-2. Six corresponding polypeptides of these 2 serotypes had different electrophoretic mobilities, indicating that these corresponding polypeptides differ in their molecular weights. A genetic relationship was not determined between the 2 EHDV serogroups and the blue-tongue serogroup viruses, using oligonucleotides mapping.

Animals↗
PubMed1985

Molecular epidemiology of two US orbiviruses: bluetongue virus and epizootic hemorrhagic disease virus.

The degree of relatedness between 4 US serotypes (10, 11, 13 and 17) of bluetongue (BT) virus (BTV) were determined by both oligonucleotide fingerprint analyses of double-stranded (ds) RNA as well as tryptic peptide analyses of viral coded polypeptides. Similar studies were undertaken using different alternate isolates of particular serotypes as well as 2 serotypes (1 and 2) of epizootic hemorrhagic disease (EHD) virus (EHDV), a closely related serogroup of orbiviruses. The results indicate that all BT viruses are genetically related to each other and although EHDV-1 and EHDV-2 originated from a common gene pool, the 2 serogroups, EHDV and BTV, are not genetically interactive.

Bluetongue virus↗